52 Essays in Biochemistry 



needed to mediate the metabolic effect of irradiation. We must admit 

 that we know of no evidence for the existence of a radiation-sensitive 

 cofactor of induction. The hypothesis is frankly rooted in heuristic 

 opportunism: nothing much could be done chemically with an hy- 

 pothesis postulating some subtle change in a macromolecule during 

 starvation. 



We therefore started a search for a metabolite which, upon irradia- 

 tion, might act as an inducer on unirradiated E. coli K 12; either in log- 

 arithmic growth phase, or starving. We irradiated strongly a variety 

 of metabolites (purines, pyrimidines, nucleosides, nucleotides, vitamins, 

 and coenzymes) and then incubated E. coli K ]2 in logarithmic growth 

 phase with the irradiation products. We tested the irradiation prod- 

 ucts for their toxic effects on the organisms by counting surviving 

 cells, and for their inducing effect by assaying on a sensitive strain 

 for an increase in the normal background of infectious centers. 



We found that several metabolites upon strong irradiation are con- 

 verted into products toxic to the bacteria. Some of these had been 

 described in the literature; some had not. But of all the irradiated 

 metabolites only leucovorin, and its derivative anhydroleucovorin, 

 acted as inducers. Such an experiment was performed as follows: 

 Five hundred micrograms of anhydroleucovorin or calcium leucovorin 

 tetrahydrate per milliliter in the usual synthetic medium was irradiated 

 in a 10-ml. lot in a quartz petri dish for 15 hours with a 15-watt 

 G.E. germicidal lamp. E. coli K 12 in logarithmic growth phase was 

 centrifuged, and the cell clump was resuspended in the irradiated 

 medium to a concentration of about 10 8 cells per milliliter. The culture 

 was then incubated in the dark for 50 minutes, and then appropriate 

 plating was performed for the determination of surviving colony 

 formers and of infectious centers. 15 In Fig. 9 the photographs of the 

 result of such an experiment along with that of an untreated control 

 at twice the concentration are given. 



The irradiation-elicited inducing potency resides in the pteridine 

 moiety of leucovorin, since 2-amino-4-hydroxy-5-formyl-6-methyl- 

 5,6,7,8-tetrahydropteridine 16 (by analogy to folic acid a plausible first 

 cleavage product of leucovorin 17 ) is converted by 5 hours of irradiation 

 into an inducer, but para-aminobenzoylglutamic acid is not. 



In Table 1 characteristic data are presented. It should be empha- 

 sized that the induction by the irradiated products is only partial. 

 Full induction, by 100 seconds irradiation, would induce, under these 

 conditions, about 60% of the cells. No higher induction could, as yet, 



